Separation and purification method of vanillin fermentation liquor

Abstract

The invention discloses a separation and purification method of vanillin fermentation liquor, and belongs to the technical field of biochemical engineering. The method comprises the following steps: microfiltration, adsorption and impurity removal, elution, concentration, heat preservation and liquid separation, cooling and crystallization, and filtration and drying. The method is simple and convenient in process, large in treatment capacity, less in generation of industrial three wastes, green and environment-friendly, the obtained vanillin is pure white crystal powder, the purity can reach 99.5% or above, and the comprehensive yield can reach 88% or above.

Description

Technical Field

[0001] The invention relates to a method for separating and purifying vanillin fermentation broth, and belongs to the technical field of biochemical engineering. Background Art

[0002] Vanillin, also known as vanillin, has a chemical name of 3-methoxy-4-hydroxybenzaldehyde. It is an organic compound extracted from vanilla beans of the orchid family. It is a white to slightly yellow crystalline powder. White vanillin with a purity of >99.5% is of better quality. Vanillin has the aroma of vanilla beans and a strong milky aroma. It has the effect of enhancing and fixing the aroma. It is widely used in the cosmetics, tobacco, cakes, candy, baked food and other industries. It is one of the synthetic flavors with the largest output in the world.

[0003] There are many methods for producing vanillin. However, the prices of vanillin obtained by different production methods vary greatly. The differences are as follows: (1) Vanillin is prepared by plant extraction, and the market price is 1,000 US dollars / kg; (2) Chemical synthesis method, according to the starting synthetic raw materials, can be divided into several methods such as guaiacol method, lignin method, safrole method, eugenol method, p-hydroxybenzaldehyde, 4-methylguaiacol method, p-cresol method, etc. At present, the industry mainly uses the guaiacol method, and the market price is 10 US dollars / kg. (3) Microbial fermentation method is used to transform and produce vanillin, and then separated and purified, and the market price is 500 US dollars / kg. At present, the annual demand for vanillin in the world is 25,000 tons, but the efficiency of extracting vanillin from plants is too low and the cost is too high. For example, the vanillin content in dry vanilla beans is only 15-20g / kg, and the natural vanillin extracted from vanilla beans each year is less than 20 tons, which is far from meeting the market demand. Although chemical synthesis of vanillin is low-cost and large-scale, it is easy to cause environmental pollution and cannot meet people's requirements for natural food. The process of producing natural vanillin by microbial fermentation is simple, with high yield, low energy consumption, low pollution, and safe and reliable products. The US FDA stipulates that "natural flavors must be products obtained from raw materials from plants or animals through physical, enzymatic or microbial processing." Therefore, synthesizing vanillin by microbial fermentation and widely using it in industrial production is a future development trend.

[0004] However, there are still many problems in the currently disclosed extraction and purification processes of vanillin fermentation broth. For example, in the invention patent CN103709021A, the vanillin in the fermentation broth is extracted by an extraction process using easily polluted organic solvents such as n-hexane, ethyl acetate and petroleum ether. In the process, a large amount of acid / base is used for pH adjustment, which will produce a large amount of waste hydrochloric acid, and the purity of the final product is also low; in the embodiments of the invention patents CN102321563A and CN101165168A, the resin adsorption method is used alone for extraction, and the yield of the product is only 65%. The quality is also deviated; in the invention patents CN104955759A and CN110312700B, the natural vanillin crude product that has been crudely extracted is subjected to secondary processing by vacuum distillation to obtain high-quality vanillin, but this method has very high requirements on the equipment, because the boiling point and melting point of vanillin are very high, and it needs to be evaporated under ultra-high vacuum and high temperature conditions, which is difficult to achieve with ordinary equipment, and the product is easy to deteriorate under high temperature conditions, and the yield is low. In the patent, the loss rate of the single step of vacuum evaporation is as high as about 15%.

[0005] Through patent research, it is found that the extraction difficulty of vanillin fermentation broth produced by fermentation is still very large, mainly because the market has very high quality requirements for natural vanillin products (white crystals, purity> 99.5%), and the commonly used extraction and resin processes cannot extract and purify high-quality vanillin from the fermentation broth, and some toxic and harmful organic solvents are often used in the existing extraction process, which affects food safety and environmental protection; in addition, if the vacuum evaporation process is adopted, the equipment investment is very large, the energy consumption is high, and the yield is low, and the final production cost will also be greatly increased. Therefore, the current extraction and purification process for vanillin fermentation broth is generally more complicated, and some toxic and harmful reagents or organic solvents are often used in the process to improve the purification efficiency, the environmental pressure is large, and the product quality is deviated, or a high-energy consumption and high-cost process such as vacuum evaporation is used. The existing technology lacks a vanillin purification method that can ensure the high quality of vanillin under the premise of simple process, low cost and no pollution, so that the obtained vanillin purity is above 99.5% and the product is white. Summary of the invention

[0006] [Technical issues]

[0007] The technical problem to be solved by the present invention is that the existing method for separating and purifying vanillin from fermentation broth has low product quality, high energy consumption, large equipment investment, low operability, and great environmental pressure, and the obtained vanillin product has low purity and poor appearance.

[0008] [Technical solution]

[0009] The invention provides a method for separating and purifying vanillin fermentation broth. The method has simple process, large processing capacity and less generation of three industrial wastes. The obtained vanillin is pure white crystalline powder with a purity of up to 99.5% and a recovery rate of more than 88%.

[0010] The method comprises the following steps:

[0011] S1, microfiltration

[0012] The vanillin fermentation broth was sterilized by microfiltration using a ceramic membrane, and the ceramic membrane dialysate was collected;

[0013] The fermentation broth is fermented with a recombinant Corynebacterium glutamicum capable of synthesizing vanillin as a fermentation strain, and with ferulic acid, glucose, corn steep liquor and trace elements as substrates. The main components of the fermentation broth are vanillin, ferulic acid, vanillic acid, pigment, sugar, salt, protein, polypeptide and bacterial cells.

[0014] S2, adsorption and impurity removal

[0015] The vanillin in the dialyzed clear solution of step S1 is adsorbed on the resin column by using a macroporous adsorption resin, and the impurities such as substrate, residual sugar, salt, part of pigment and protein in the fermentation broth are removed along with the dialyzed solution;

[0016] S3, elution

[0017] The vanillin adsorbed on the macroporous resin in step S2 is eluted with an organic solvent aqueous solution, and the feed liquid is collected when vanillin begins to be detected in the eluate, and the elution is terminated when vanillic acid is detected in the eluate, and the eluate is collected;

[0018] S4, evaporation of solvent

[0019] Distilling off the organic solvent in the eluate of step S3 to obtain a vanillin concentrate;

[0020] S5, Concentration

[0021] The vanillin concentrate in step S4 is concentrated by reduced pressure evaporation at 50-80° C., and when obvious stratification occurs in the system, the concentration is stopped to obtain a stratified liquid; the concentration of vanillin in the stratified liquid is ≥40 g / L;

[0022] S6, heat preservation and liquid separation

[0023] The layered liquid in step S5 is kept at 50-80° C. for at least 10 min and then separated, and the lower organic phase and the upper aqueous phase are collected respectively;

[0024] S7, cooling, crystallization and filtration and drying

[0025] The upper aqueous phase in step S6 is transferred to a crystallization tank for cooling and crystallization, the crystals are filtered to obtain a filter cake, and the filter cake is washed and dried to obtain pure vanillin.

[0026] In one embodiment of the present invention, in step S5, the concentration of vanillin in the layered liquid is ≥50 g / L.

[0027] In one embodiment of the present invention, in step S6, the temperature is kept at 50-70°C; the temperature of the keeping in step S6 is the same as the temperature of the concentration in step S5.

[0028] In one embodiment of the present invention, in step S6, the standing time is 10 to 60 minutes.

[0029] In one embodiment of the present invention, the pore size of the ceramic membrane used in step S1 is between 50 nm and 200 nm.

[0030] In one embodiment of the present invention, the macroporous resin in step S2 is a non-polar macroporous resin, a weakly polar macroporous resin or a polar macroporous resin, such as LX-8G, LX-3020, LX-T83 and LX-67.

[0031] In one embodiment of the present invention, the processing capacity of the macroporous resin in step S2 is 1 BV to 20 BV, and the loading flow rate is 1 BV / h to 10 BV / h.

[0032] In one embodiment of the present invention, after the injection of the flow-through liquid in step S2 is completed, the resin needs to be washed with pure water to remove the impurities remaining in the resin column. The end point of the washing is determined by the conductivity of the water washing liquid, and the end point conductivity is <100us / cm.

[0033] In one embodiment of the present invention, the organic solvent in step S3 is methanol or ethanol; the concentration of the organic solvent is 20% to 60%; and the elution flow rate is 0.5 BV / h to 5 BV / h.

[0034] In one embodiment of the present invention, the elution process in step S3 is monitored by thin layer chromatography, and the collection of the feed liquid begins when the spot plate shows that vanillin is eluted, and the elution ends when the spot plate shows that vanillic acid is eluted.

[0035] In one embodiment of the present invention, the method of evaporating the solvent in step S4 is atmospheric distillation, and the boiling point of the steam is monitored during the evaporation process. When the boiling point of the steam is greater than 99.5° C. or reaches 100° C., the evaporation of the solvent is stopped.

[0036] In one embodiment of the present invention, the concentration method in step S5 is reduced pressure concentration, the vacuum degree is less than -0.08Mpa, and the evaporation temperature is greater than 50°C.

[0037] In one embodiment of the present invention, the crystallization method in step S7 is gradient cooling crystallization, the cooling gradient is 1 to 10 min / °C, the cooling end temperature is 0 to 15°C, and the crystallization time is 4 to 20 hours.

[0038] In one embodiment of the present invention, the solvent used for washing the filter cake in step S7 is pure water.

[0039] In one embodiment of the present invention, the drying in step S7 is vacuum drying, the drying temperature is 30 to 70° C., the vacuum degree is less than -0.08 MPa, and the drying time is 12 to 24 hours.

[0040] The invention also discloses application of the method in preparing vanillin or a product containing vanillin.

[0041] [Beneficial Effects]

[0042] 1. The vanillin extraction and purification method of the present invention solves the problems of difficulty in purifying vanillin produced by microbial fermentation and low quality of purified products. The extraction process is simple to operate, has few steps and high purification efficiency. The obtained vanillin is a pure white crystalline powder with a purity of up to 99.5% and a yield of more than 88%.

[0043] 2. The method of the present invention has low energy consumption, no pollution and low equipment investment, and the organic solvents used are only ethanol or methanol, both of which are environmentally friendly and non-toxic organic solvents, which greatly improves product competitiveness and production profits.

[0044] 3. The present invention utilizes the property that vanillin dissolves saturatedly in water at a certain concentration and temperature and then separates into layers, and uses a very small amount of separated vanillin as an extractant to extract impurities such as pigments and vanillic acid remaining in the system, and this part of the vanillin can also be recycled and reused, thereby solving the problem of yellowish color and poor quality of vanillin products in an extremely simple way and reducing the purification cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a process flow chart.

[0046] Figure 2 This is a diagram showing the effect of the stratification of the liquid after standing in step S6 of Example 1 (the lower layer in the figure is the vanillin phase, and the upper layer is the water phase). DETAILED DESCRIPTION

[0047] The fermentation broth used in the following examples is fermented with a recombinant Corynebacterium glutamicum capable of synthesizing vanillin as the fermentation strain, and with ferulic acid, glucose, corn steep liquor and trace elements as substrates. The main components of the fermentation broth are vanillin, vanillic acid, ferulic acid, pigment, sugar, salt, protein, polypeptide and bacterial cells. The composition of the vanillin fermentation broth is shown in Table 1 after testing:

[0048] Table 1

[0049] sample Vanillin content g / L Vanillic acid content g / L Ferulic acid content g / L Residual sugar % Conductivity ms / cm Sample 1 10.5 0.3 0.03 3.05 13.93 Sample 2 10.2 0.29 0.07 2.63 14.35 Sample 3 10.8 0.33 0.02 2.82 14.81

[0050] Vanillin thin layer detection method:

[0051] The developing solvent is ethanol: dichloromethane: acetic acid = 1:10:0.5, and the display wavelength is ultraviolet 254nm.

[0052] Detection method of pure vanillin:

[0053] Detection method of vanillin GB 1886.16-2015 "National Food Safety Standard Food Additive Vanillin"

[0054] GC detection of vanillin, column: capillary column, length 25m~50m, inner diameter 0.2mm; stationary phase: 100% dimethylpolysiloxane; film thickness: 0.25μm; chromatographic oven temperature: 170℃ constant temperature; injection port temperature: 240℃; detector temperature 240℃; detector: hydrogen flame ionization detector; carrier gas: nitrogen; carrier gas flow rate: 1mL / min; injection volume: about 0.2μL, split ratio: 100:1.

[0055] Sample preparation: Weigh 1 g of the sample and dissolve it in 10 mL of 95% (volume fraction) ethanol, shake well and set aside.

[0056] The ceramic membrane circulation tank in the following examples was purchased from Hefei Xinda Membrane Technology Co., Ltd., model number is XD-T2-1000;

[0057] The macroporous resins in the following examples were purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0058] Example 1

[0059] The method for extracting and separating vanillin from fermentation broth of the present invention comprises the following steps:

[0060] S1, microfiltration

[0061] Take 20L of vanillin fermentation liquid in sample 1 and add it into the circulation tank in the ceramic membrane. Turn on the motor, adjust the feed pressure to 0.2MPa, collect and combine the dialysate and water top feed liquid, totaling 25L.

[0062] S2, adsorption and impurity removal

[0063] 3L of regenerated LX-3020 resin was loaded into the resin column, and the dialysate in S1 was passed through the column at a flow rate of 6L / h for adsorption. The vanillin in the dialysate was adsorbed on the resin column, and the substrate, residual sugar, salt, some pigments and proteins in the fermentation broth were removed with the dialysate. When all the feed liquid was loaded, the impurities remaining in the resin column were washed with pure water. The water washing flow rate was 6L / h, and about 6L of water was used. The conductivity at the end of the water washing was 88us / cm (or <100us / cm), and no vanillin residue was detected in the flow liquid and the washing liquid.

[0064] S3, elution

[0065] Prepare some 50% ethanol-water solution for later use, then elute at a flow rate of 1 BV / h. Use thin layer chromatography for spot plate monitoring during the process. Start collecting the eluate when vanillin is discharged, and stop eluting when vanillic acid is discharged. Collect 15.8 L of eluate in total.

[0066] S4, evaporation of solvent

[0067] The S3 eluate was transferred in batches to a 10L glass evaporator for normal pressure solvent evaporation. The boiling point of the vapor fraction was recorded during the process. When the boiling point reached 100°C, the solvent evaporation was stopped. At this time, the volume of the concentrated liquid was 6.95L. The vanillin content in the sampled liquid was 27.8g / L.

[0068] S5, Concentration

[0069] The evaporation was started, and the liquid in S4 was further concentrated by reduced pressure evaporation. The kettle temperature was 70°C and the vacuum degree was -0.08Mpa. During the concentration process, the solubility of vanillin in water reached saturation and precipitated in liquid form, and it would separate from the water phase. The separation phenomenon was as follows: Figure 2 As shown (the upper layer is a saturated aqueous solution of vanillin, and the lower layer is a vanillin phase in which some water, pigments and impurities are dissolved), when the volume of the feed liquid is about 2.8L, after calculation, the concentration of vanillin in the system is about 69g / L, and evaporation and concentration is stopped.

[0070] S6, heat preservation and separation (aqueous phase extraction)

[0071] The separated liquid in S5 was kept at 70°C for 30 min and then separated, and the lower organic phase and the upper aqueous phase were collected respectively;

[0072] S7, cooling, crystallization and filtration and drying

[0073] The upper aqueous phase in step S6 was transferred to a crystallization tank to start cooling and crystallization, and then slowly cooled to 4°C at a cooling rate of 5 min / °C, kept warm for 2 hours, the crystals were filtered, and washed with pure water. After washing, the wet crystals were placed in a vacuum drying oven for vacuum drying, and dried at 60°C, -0.09 MPa vacuum for 24 hours. The vanillin product was taken out, weighing 162.2 g, and the GC detection content was 99.6%. The mother liquor volume was 2.6 L, and the vanillin content in the mother liquor was 3.27 g / L The direct recovery rate is 77.24%. After calculation, the mother liquor contains 8.5g of vanillin. In addition, through material balance, the total mass of vanillin separated during separation is: 193.2g-162.2g-8.5g=22.5g. Since the applied process is the same as the main purification process, the applied yield is also calculated according to 77.24%. Finally, about (22.5+8.5)*77.24%=23.94g of qualified products can be applied, and the final comprehensive yield is about 88.64%.

[0074] After testing, the technical effects of the present invention can be achieved by replacing the macroporous adsorption resin LX-3020 in step S2 with LX-8G, LX-T83 or LX-67, adjusting the organic solvent concentration in step S3 to 20% to 60%, and adjusting the pore size of the ceramic membrane in step S1 to 50nm to 200nm.

[0075] Example 2

[0076] The method for extracting and separating vanillin from fermentation broth of the present invention comprises the following steps:

[0077] S1, microfiltration

[0078] Take 20L of vanillin fermentation liquid in sample 2 and add it into the circulation tank in the ceramic membrane. Turn on the motor, adjust the feed pressure to 0.2MPa, collect and combine the dialysate and water top feed liquid, totaling 25.4L.

[0079] S2, adsorption and impurity removal

[0080] 3L of regenerated LX-3020 resin was loaded into the resin column, and the dialysate in S1 was passed through the column at a flow rate of 6L / h for adsorption. After all the feed liquid was loaded, the impurities remaining in the resin column were washed with pure water at a flow rate of 6L / h, using about 6L of water. The conductivity at the end of the water washing was 94us / cm, and no vanillin residue was detected in the flow liquid and the washing liquid.

[0081] S3, elution

[0082] Prepare some 50% methanol aqueous solution for later use, then elute at a flow rate of 1 BV / h. Use thin layer chromatography for spot plate monitoring during the process. Start collecting the eluate when vanillin is discharged, and stop eluting when vanillic acid is discharged. Collect 14.9 L of eluate in total.

[0083] S4, evaporation of solvent

[0084] The S3 eluate was transferred in batches to a 10L glass evaporator for normal pressure solvent evaporation. The boiling point of the vapor fraction was recorded during the process. When the boiling point reached 100°C, the solvent evaporation was stopped. At this time, the volume of the concentrated liquid was 7.2L. The vanillin content in the sampled liquid was 26.63g / L.

[0085] S5, Concentration

[0086] The evaporation was started, and the feed liquid in S4 was further concentrated by reduced pressure evaporation. The kettle temperature was 70° C. and the vacuum degree was -0.08 MPa. During the concentration process, the solubility of vanillin in water reached saturation and precipitated in liquid form, and it would separate from the water phase. The separation phenomenon was similar to the separation phenomenon in step S5 of Example 1. When the volume of the feed liquid was about 2.75 L, the concentration of vanillin in the system was calculated to be about 69.72 g / L, and the evaporation concentration was stopped.

[0087] S6, heat preservation and liquid separation

[0088] The separated liquid in S5 was kept at 70°C for 40 min and then separated, and the lower organic phase and the upper aqueous phase were collected respectively;

[0089] S7, cooling, crystallization and filtration and drying

[0090] The upper aqueous phase in step S6 was transferred to a crystallization tank to start cooling and crystallization, and then slowly cooled to 4°C at a cooling rate of 5 min / °C, kept warm for 2 hours, the crystals were filtered, and washed with pure water. After washing, the wet crystals were placed in a vacuum drying oven for vacuum drying, and dried at 60°C, -0.09 MPa vacuum for 24 hours. The vanillin product was taken out, weighing 160.9 g, and the GC detection content was 99.7%. The volume of the mother liquor was 2.6 L, and the vanillin content in the mother liquor was 3.31 g / L, the direct recovery yield is 76.62%. After calculation, the mother liquor contains 8.6g of vanillin. In addition, through material balance, the total mass of vanillin separated during separation is: 191.7g-160.9g-8.6g=22.2g. Since the applied process is the same as the main purification process, the applied yield is also calculated as 76.62%. Finally, about (22.2+8.6)*76.62%=23.6g of qualified products can be applied, and the final comprehensive yield is about 87.86%.

[0091] Example 3

[0092] The method for extracting and separating vanillin from fermentation broth of the present invention comprises the following steps:

[0093] S1, microfiltration

[0094] Take 20L of vanillin fermentation liquid in sample 2 and add it into the circulation tank in the ceramic membrane. Turn on the motor, adjust the feed pressure to 0.2MPa, collect and combine the dialysate and water top feed liquid, totaling 25.5L.

[0095] S2, adsorption and impurity removal

[0096] 3L of regenerated LX-8G resin was loaded into the resin column, and the dialysate in S1 was passed through the column at a flow rate of 6L / h for adsorption. After all the feed liquid was loaded, the impurities remaining in the resin column were washed with pure water at a flow rate of 6L / h, using about 6L of water. The conductivity at the end of the water washing was 79us / cm, and no vanillin residue was detected in the flow liquid and the washing liquid.

[0097] S3, elution

[0098] Prepare some 50% methanol-water solution for later use, then elute at a flow rate of 1 BV / h. Use thin layer chromatography for spot plate monitoring during the process. Start collecting the eluate when vanillin is discharged, and stop eluting when vanillic acid is discharged. Collect 17.8 L of eluate in total.

[0099] S4, evaporation of solvent

[0100] The S3 eluate was transferred in batches to a 10L glass evaporator for normal pressure solvent evaporation. The boiling point of the vapor fraction was recorded during the process. When the boiling point reached 100°C, the solvent evaporation was stopped. At this time, the volume of the concentrated liquid was 8.1L. The vanillin content in the sampled liquid was 23.55 g / L.

[0101] S5, Concentration

[0102] The evaporation was started, and the feed liquid in S4 was further concentrated by reduced pressure evaporation. The kettle temperature was 70° C. and the vacuum degree was -0.08 MPa. During the concentration process, the solubility of vanillin in water reached saturation and precipitated in liquid form, and it would separate from the water phase. The separation phenomenon was similar to the separation phenomenon in step S5 of Example 1. When the volume of the feed liquid was about 2.92 L, the concentration of vanillin in the system was calculated to be about 65.33 g / L, and the evaporation concentration was stopped.

[0103] S6, heat preservation and liquid separation

[0104] The separated liquid in S5 was kept at 70°C for 50 min and then separated, and the lower organic phase and the upper aqueous phase were collected respectively;

[0105] S7, cooling, crystallization and filtration and drying

[0106] The upper aqueous phase in step S6 was transferred to a crystallization tank to start cooling and crystallization, and then slowly cooled to 4°C at a cooling rate of 5 min / °C, kept warm for 2 hours, the crystals were filtered, and washed with pure water. After washing, the wet crystals were placed in a vacuum drying oven for vacuum drying, and dried at 60°C, -0.09 MPa vacuum for 24 hours. The vanillin product was taken out, weighing 159.4 g, with a GC content of 99.81%, a mother liquor volume of 2.76 L, and a vanillin content of 3.15 g / L in the mother liquor. The direct recovery rate is 75.9%. After calculation, the mother liquor contains 8.7g of vanillin. In addition, through material balance, the total mass of vanillin separated during separation is: 190.755g-159.4g-8.7g=22.655g. Since the applied process is the same as the main purification process, the applied yield is also calculated as 75.9%. Finally, about (22.655+8.7)*75.9%=23.8g of qualified products can be applied, and the final comprehensive yield is about 87.24%.

[0107] Comparative Example 1

[0108] This comparative example provides a method for purifying vanillin from a fermentation broth. The specific steps are the same as those in Example 1, except that the concentration temperature in step S5 and the insulation temperature in step S6 are adjusted to 40°C, 50°C, 60°C and 80°C, respectively. The experimental results are shown in Table 2:

[0109] Table 2

[0110]

[0111] As can be seen from the above table, under the condition of 40°C, due to the relatively low temperature, vanillin is difficult to separate into the aqueous phase, and vanillin directly crystallizes during the concentration process, and no qualified product is obtained; when the temperature reaches above 50°C, vanillin will separate into the aqueous phase in liquid form under high temperature / high concentration, thereby achieving purification of the product. With the increase of concentration and insulation temperature, the product yield steadily increases, but when the temperature is higher than 80°C, it will affect the product quality.

[0112] Comparative Example 2

[0113] This comparative example provides a method for purifying vanillin from a fermentation broth. The specific steps are the same as those in Example 1, except that the concentrations at the end points in step S5 are 31 g / L, 40.5 g / L, 50.9 g / L and 70.3 g / L, respectively. The experimental results are shown in Table 3:

[0114] Table 3

[0115] Experiment Name Concentration end point concentration g / L purity% Single step yield% Comprehensive yield% Product color Comparative Example 2 31 / / / / Comparative Example 2 40.5 98.6 79.5 90.13 Yellowish Comparative Example 2 50.9 99.1 78.83 89.87 white Example 1 69 99.6 77.24 88.64 white Comparative Example 2 70.3 99.8 73.89 84.22 white

[0116] It can be seen from the above table that if the concentration is too low, there will be no stratification. When the concentration is higher than 40g / L, stratification will begin. Later, as the concentration at the concentration end point increases, the amount of vanillin in the stratification will increase, the impurities carried away will increase, the product quality will improve, but the yield will be lost.

[0117] Comparative Example 3

[0118] This comparative example provides a method for purifying vanillin from a fermentation broth. The specific steps are the same as those of Example 1, except that in step S6, the stratification is not allowed to stand, and the crystallization is directly stirred and cooled, or other extractants are used for extraction, decolorization and impurity removal. The amount of the extractant is 1 / 2 of the volume of the aqueous phase. The experimental results are shown in Table 4:

[0119] Table 4

[0120] Experiment Name Extraction method purity% Single step yield% Comprehensive yield% Product color Example 1 Aqueous extraction 99.6 77.24 88.64 white Comparative Example 3 No extraction 97.7 86.81 91.41 yellow Comparative Example 3 Toluene extraction 98.95 54.02 73.62 Light yellow Comparative Example 3 Ethyl acetate extraction 98.13 62.64 82.34 Light yellow Comparative Example 3 Dichloromethane 99.05 65.53 84.22 Light yellow

[0121] The calculation method of organic solvent extraction yield is to take toluene extraction as an example: after the water phase is crystallized, it is dried and weighed to obtain 114.65g of product, with a content of 98.95%, a net content of vanillin of 113.44g, and a direct yield of 54.02%; the volume of the mother liquor is 2.64L, the content is 3.4g / L, and the mother liquor contains 8.98g of vanillin; the volume of the toluene phase is 1.28L, and the content is 55.3g / L. The mother liquor of the water phase crystallization can be directly applied, and the vanillin in the toluene phase needs to be concentrated and crystallized to obtain the crude vanillin After that, the concentrated crystallization yield of vanillin in the toluene phase is controlled at more than 95%. The amount of vanillin that can be applied is about 1.28L*55.3g / L*95%=67.24g, plus 8.98g vanillin in the mother liquor, the amount of vanillin that needs to be applied is about 76.22g, and the application yield is calculated as 54.02%. Finally, about (67.24+8.98)*54.02%=41.18g of qualified products can be applied, and the final comprehensive yield is about 73.62%.

[0122] Comparative Example 4

[0123] This comparative example provides a method for purifying vanillin from a fermentation broth. The specific steps are the same as those in Example 1, except that in the implementation process of Comparative Example 3, reference is made to Figure 1 The process flow is as follows: the lower layer of vanillin material after stratification in step S6 of Example 1 is applied to step S2 of comparative example 3, and is combined with the feed liquid of step S1 of comparative example 3 to pass through the column; the crystallization mother liquor of step S7 of Example 1 is applied to step S5 of comparative example 3, and is combined with the feed liquid of step S4 of comparative example 3 to be concentrated. The final experimental results are shown in Table 5 below:

[0124] Table 5

[0125] Experiment Name purity% Single step yield% Comprehensive yield% Product color Example 1 99.6 77.24 88.64 (theoretical) white Comparative Example 3 99.68 88.06 (actual) / white

[0126] As can be seen from the above table, Comparative Example 3 is an application verification of the materials in Example 1. On the one hand, the product quality is not affected by the application, and on the other hand, the comprehensive yield in Example 1 is also theoretically verified and has certain rationality.

[0127] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for separating and purifying vanillin, characterized in that: The following steps are involved: S1, microfiltration The feed solution containing vanillin is sterilized by microfiltration, and the dialysate is collected; S2, adsorption and impurity removal The dialysate in step S1 is adsorbed by a macroporous adsorption resin, and after the adsorption is completed, the column is washed to obtain a macroporous adsorption resin that adsorbs vanillin; S3, elution Using an organic solvent aqueous solution to elute the macroporous adsorption resin adsorbing vanillin in step S2, and collecting the eluate; S4, evaporation of solvent Distilling off the organic solvent in the eluate collected in step S3 to obtain a vanillin concentrate; S5, Concentration The vanillin concentrate in step S4 is concentrated by reduced pressure evaporation at 50-80° C., and when obvious stratification occurs in the system, the concentration is stopped to obtain a stratified liquid; the concentration of vanillin in the stratified liquid is ≥40 g / L; S6, heat preservation and liquid separation The layered liquid in step S5 is kept at 50-80° C. for at least 10 min and then separated, and the lower organic phase and the upper aqueous phase are collected respectively; S7, cooling, crystallization and filtration and drying The upper aqueous phase in step S6 is cooled and crystallized, filtered, washed and dried to obtain pure vanillin.

2. The method according to claim 1, characterized in that In step S5, the concentration of vanillin in the layered liquid is ≥50 g / L.

3. The method according to claim 1, characterized in that In step S6, the temperature is kept at 50-70°C; the temperature of the keeping in step S6 is the same as the temperature of the concentration in step S5.

4. The method according to claim 1, characterized in that: In step S6, the standing time is 10 to 60 minutes.

5. The method according to claim 1, characterized in that In step S3, the organic solvent is methanol or ethanol; in the organic solvent aqueous solution, the organic solvent concentration is 20% to 60%; and the elution flow rate is 0.5 BV / h to 5 BV / h.

6. The method according to claim 1, characterized in that In step S1, microfiltration is performed using a ceramic membrane, wherein the pore size of the ceramic membrane is 50 nm to 200 nm.

7. The method according to claim 1, characterized in that The macroporous adsorption resin in step S2 includes non-polar macroporous resin, weak polar macroporous resin or polar macroporous resin; preferably, the macroporous adsorption resin includes LX-8G, LX-3020, LX-T83 or LX-67.

8. The method according to claim 1, characterized in that The processing capacity of the macroporous resin in step S2 is 1 BV to 20 BV, and the loading flow rate is 1 BV / h to 10 BV / h.

9. The method according to claim 1, characterized in that: In step S2, the column is washed with water. After the column is washed, the conductivity of the water washing liquid is less than 100 us / cm.

10. Use of the method according to any one of claims 1 to 9 in the preparation of vanillin or a product containing vanillin.

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